Genetic markers for discrimination and detection of viral hemorrhagic septicemia virus causing infectious aquatic organism diseases, and method of discriminating and detecting the virus using the same

ABSTRACT

Genetic markers are described for discriminating or detecting viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same is disclosed, in which the method includes selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish are infected with the viral type by analyzing the obtained melting curve to determine a melting temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S. patentapplication Ser. No. 15/740,475 filed Dec. 28, 2017, which in turn is aU.S. national phase under the provisions of 35 U.S.C. § 371 ofInternational Patent Application No. PCT/KR2016/009373 filed Aug. 24,2016, which in turn claims priority under 35 U.S.C. § 119 of KoreanPatent Application No. 10-2016-0005611 filed Jan. 15, 2016. Thedisclosures of U.S. patent application Ser. No. 15/740,475,International Patent Application No. PCT/KR2016/009373, and 35 U.S.C. §119 of Korean Patent Application No. 10-2016-0005611 are herebyincorporated herein by reference in their respective entireties, for allpurposes.

TECHNICAL FIELD

The present invention relates to genetic markers for discrimination anddetection of viruses causing infectious aquatic organism diseases, and amethod of discriminating and detecting the viruses using the same. Morespecifically, the present invention relates to a method fordiscriminating or detecting viruses causing infectious aquatic organismdiseases, the method comprising: selecting and amplifying a DNAnucleotide sequence encoding a gene specific for viral hemorrhagicsepticemia virus (VHSV), red sea bream iridovirus (RSIV) or infectiousspleen and kidney necrosis virus (ISKNV), which is a virus causing redsea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing apeptide nucleic acid (PNA) that specifically recognizes theamplification product; controlling the temperature of the hybridizationproduct to obtain a temperature-dependent melting curve; anddiscriminating the viral type or detecting whether or not fish would beinfected with the viral type by analyzing the obtained melting curve todetermine a melting temperature.

BACKGROUND ART

Among recent methods for diagnosis of infectious diseases, diagnosticmethods and kits based on molecular diagnosis have been developed. Amongthese methods, a method has been mainly used, which comprises performinggeneral polymerase chain reaction (PCR) to obtain an amplificationproduct, and then either analyzing the amplification product byelectrophoresis, or analyzing the amplification by real-time PCR using afluorescent probe or SYBR green.

Particularly, among infectious aquatic organism diseases, viralhemorrhagic septicemia virus (VHSV), red sea bream iridovirus(RSIV)/infectious spleen and kidney necrosis virus (ISKNV), or Koiherpesvirus (KHV), is classified as a nationally notifiable infectiousdisease. For the purpose of detection of this virus, according to thestandards set forth in the Aquatic Animal Health Code of the WorldOrganization for Animal Health (OIE), conventional PCR is performed toobtain an amplification product, and the amplification product isanalyzed by electrophoresis, after which the amplification product iscloned, and the nucleotide sequence of the purified plasmid DNA isanalyzed by a process such as Sanger sequencing. Thus, there aredisadvantages in that the procedures are complex and analysis fordisease diagnosis is time-consuming.

For molecular diagnosis of the fish virus VHSV, various techniques forgenetic diagnosis have been developed, including RT-PCR and real-timeRT-PCR, which enables diagnosis with high accuracy and sensitivitywithin a short time. For VHSV diagnosis, a conventional RT-PCR processcomposed of one step or two steps is used according to the OIEstandards. Particularly, as genetic variants of the virus have recentlybeen cyclically found in North America, Asia, Europe and Atlantic areas,studies on the epidemiological dynamics of the causative virus have beenconducted, and genotype analysis based on sequencing of an amplificationproduct, which is performed after performing PCR according to the OIEstandards in order to obtain accurate results in molecular diagnosis,has been of increasing importance. In recent years, techniques such asLAMP (loop-mediated isothermal amplification) for simple genotypeanalysis have been developed, but these techniques are not recommendedby the OIE, since there is a limit to detection of viruses havingvarious genotypes.

For the purpose of detection of RSIV or ISKNV, which is a virus causingred sea bream iridovirus disease, a staining technique based on a tissuesmear sample, and serological diagnostic methods such as IFAT based onMAb, are used. For molecular diagnosis, conventional PCR is performedusing two kinds of primers according to the OIE standards. Thediagnostic methods recommended by the OIE are methods for diagnosis ofred sea bream iridovirus disease (RSIVD). Among them, the use of the“OIE protocol 1 (OIE 1)” PCR method makes it possible to diagnose thered sea bream iridovirus disease without discrimination between RSIV andISKNV, and the use of the “OIE protocol 2 (OIE 4)” PCR method makes itpossible to discriminate between RSIV and ISKNV, which are virusescausing the red sea bream iridovirus disease, by specifically amplifyingonly RSIV without amplifying ISKNV. Thus, there is a disadvantage inthat two conventional PCR steps according to the OIE standards should beperformed for diagnosis of red sea bream iridovirus disease and foraccurate identification of a virus causing the disease.

For the purpose of molecular diagnosis of Koi herpesvirus (KHV), twoconventional PCR methods (OIE, 2014) found to be most sensitive for KHVdiagnosis among a variety of disclosed PCR methods are performed. Thefirst method is a method using “Bercovier TK primers” developed byBercovier et al. in 2005, and the second method is a method developed byYuasa et al (Gray Sph primers/Yuasa modification). Although diagnosticmethods such as real-time PCR, which have higher sensitivity thanconventional PCR methods, are frequently performed in many diagnosticlaboratories, the above-mentioned two PCR methods are most frequentlyperformed in order to avoid contamination during sample preparation andPCR processes. At present, for diagnosis and identification of thenationally notifiable infectious diseases, conventional PCR methods areused as standard diagnostic methods recommended by nations.

Analysis of a PCR amplification product by electrophoresis according tothe OIE standards cannot be objectively achieved (gray zone), or averification step is performed, which includes cloning a faint PCRamplification product using a cloning vector and then re-confirming thenucleotide sequence of the amplification product by sequencing. Thisverification step is performed by analyzing the sequence of anon-specific PCR band, and has problems in that analytical proceduresare complex and time-consuming. Particularly, when conventional PCR(before determination by sequencing) indicates that an amplificationproduct is positive, the amplification product is recognized to have therisk of causing infectious diseases of aquatic organisms, and thuspreventive measures are taken. However, when sequencing performed laterindicates that the PCR result is false-positive, the reliability of thetesting laboratory can be lowered, and damage to fishery cooperativesmay also occur. For this reason, there is an urgent need for improvedmethods that diagnose nationally notifiable infectious diseases in arapid and accurate manner.

Under this technical background, the present inventors have madeextensive efforts to develop a method that discriminates fishdisease-causing viruses by determining whether or not a PCR product fordiscrimination or detection of VHSV, RSIV/ISKNV or KHV, which is a viruscausing infectious aquatic organism diseases, would bespecifically/nonspecifically amplified, without or before performing asequencing step, and that detects an individual (e.g., fish) infectedwith the causative virus. As a result, the present inventors haveidentified genetic markers for discrimination and/or detection of thetype of VHSV, RSIV/ISKNV or KHV, which is a virus causing fish diseases,and have found that when peptide nucleic acids and primers specific forthe genetic markers are used to obtain amplification and melting curveshaving different fluorescence intensities depending on the type ofvirus, fish disease-causing virus can be discriminated in a simple,rapid and accurate manner, thereby completing the present invention.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a genetic marker, aprimer and a PNA probe for discrimination or detection of VHSV,RSIV/ISKNV or KHV, which is a virus causing infectious aquatic organismdisease.

Another object of the present invention is to provide a composition anda kit for discrimination or detection of VHSV, RSIV/ISKNV or KHV, whichcomprises the above-described primer and the above-described PNA probe.

Another object of the present invention is to provide a methodcomprising: producing a single-strand genetic marker sequence fragmentusing the above-described primer; hybridizing the above-described PNAprobe to the produced genetic marker sequence fragment; and obtaining aTm value resulting from the hybridization of the PNA probe, therebydetermining the type of VHSV, RSIV/ISKNV or KHV, or detecting whether ornot an individual would be infected with the fish disease-causing virus.

Technical Solution

To achieve the above object, the present invention provides a geneticmarker for discrimination or detection of viral hemorrhagic septicemiavirus (VHSV), which is a virus causing infectious aquatic organismdisease, in which the genetic marker is represented by a nucleotidesequence of SEQ ID NO: 11.

The present invention also provides a genetic marker for discriminationor detection of Koi herpesvirus (KHV), which is a virus causinginfectious aquatic organism disease, in which the genetic marker isrepresented by a nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

The present invention also provides a primer for discrimination ordetection of viral hemorrhagic septicemia virus (VHSV), which is a viruscausing infectious aquatic organism disease, in which the primer isrepresented by a nucleotide sequence of SEQ ID NO: 6.

The present invention also provides a primer for discrimination ordetection of red sea bream iridovirus (RSIV) or infectious spleen andkidney necrosis virus (ISKNV), which is a virus causing infectiousaquatic organism disease, in which the primer is represented by anucleotide sequence of SEQ ID NO: 7.

The present invention also provides a primer for discrimination ordetection of red sea bream iridovirus (RSIV), which is a virus causinginfectious aquatic organism disease, in which the primer is representedby a nucleotide sequence of SEQ ID NO: 8.

The present invention also provides a primer for discrimination ordetection of Koi herpesvirus (KHV), which is a virus causing infectiousaquatic organism disease, in which the primer is represented by anucleotide sequence of SEQ ID NO: 9 or of SEQ ID NO: 10.

The present invention also provides a PNA probe for discrimination ordetection of viral hemorrhagic septicemia virus (VHSV), which is a viruscausing infectious aquatic organism disease, in which the PNA probe isrepresented by a nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a PNA probe for discrimination ordetection of Koi herpesvirus (KHV), which is a virus causing infectiousaquatic organism disease, in which the PNA probe is represented by anucleotide sequence of SEQ ID NO: 4 or of SEQ ID NO: 5.

The present invention also provides a composition and a kit fordiscrimination or detection of a virus causing infectious aquaticorganism disease, which comprises the above-described primer and theabove-described PNA probe.

The present invention also provides a method for discrimination ordetection of a virus causing infectious aquatic organism disease,comprising the steps of:

(a) extracting a target nucleic acid from a sample;

(b) amplifying a genetic marker nucleotide sequence for an infectiousaquatic organism disease-causing virus, contained in the target nucleicacid, by use of a conventional primer pair;

(c) producing a single-strand genetic marker sequence fragment using theamplified genetic marker nucleotide sequence as a template and theabove-described primer;

(d) hybridizing the above-described PNA probe to the producedsingle-strand genetic marker sequence fragment;

(e) obtaining a temperature-dependent melting curve while increasing thetemperature of a PNA probe-hybridized product resulting from step (d);and

(f) discriminating the viral type of the infectious aquatic organismdisease-causing virus or detecting whether or not fish would be infectedwith the viral type by analyzing the melting curve obtained in step (e)to determine a melting temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a conceptual view showing the technical characteristics of astep of obtaining an amplification curve for identifying the type ofvirus or detecting whether or not an individual would be infected withthe viral type, in a MeltingArray for verification of a PCRamplification product.

FIG. 2 is a schematic view showing a step of obtaining a melting curveby hybridization of a peptide nucleic acid probe in a method foridentifying the type of virus and a method for detecting whether or notan individual would be infected with virus.

FIG. 3 is a gene position view illustrating nucleotide sequence regionsincluded in a primer and a peptide nucleic acid probe in anamplification product obtained by the PCR method for detection of VHSVaccording to the OIE standards (SEQ ID NO: 16).

FIG. 4 is a gene position view illustrating nucleotide sequence regionsincluded in a primer and a peptide nucleic acid probe in anamplification product obtained by the “PCR protocol 1 (OIE 1)” methodfor detection of RSIV/ISKNV according to the OIE standard (SEQ ID NOS:17 and 18).

FIG. 5 is a gene position view illustrating nucleotide sequence regionsincluded in a primer and a peptide nucleic acid probe in anamplification product obtained by the “PCR protocol 2 (OIE 4)” methodfor detection of RSIV according to the OIE standards (SEQ ID NOS: 19 and20).

FIG. 6 is a gene position view illustrating nucleotide sequence regionsincluded in a primer and a peptide nucleic acid probe in anamplification product obtained by the “PCR (Bercoiver TK)” method fordetection of KHV according to the OIE standards (SEQ ID NO: 21).

FIG. 7 is a gene position view illustrating nucleotide sequence regionsincluded in a primer and a peptide nucleic acid probe in anamplification product obtained by the “PCR (Gray SpH)” method fordetection of KHV according to the OIE standards (SEQ ID NO: 22).

FIG. 8 shows amplification curve and melting curve graphs obtainedaccording to virus detection methods using the primers and peptidenucleic acid probes shown in FIGS. 1 to 7.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Generally, the nomenclatureused herein and the experiment methods, which will be described below,are those well known and commonly employed in the art.

In an example of the present invention, it was attempted to develop amethod that identifies VHSV, RSIV/ISKNV or KHV, which is an infectiousaquatic organism disease-causing virus set forth in the Aquatic AnimalHealth Code of the World Organization for Animal Health (OIE), bydetermining whether or not a PCR amplification product for a geneticmarker specific for the disease-causing virus would bespecifically/nonspecifically amplified, without or before a sequencingstep, and that detects an individual (e.g., fish) infected with thedisease-causing virus. As a result, using a marker having a specificnucleotide sequence depending the type of virus and a primer and peptidenucleic acid probe (PNA probe) for discrimination of the type of viruswhich corresponds to the marker, viruses causing infectious aquaticorganism diseases could be detected, and each virus causing infectiousaquatic organism diseases could be discriminated/detected.

More specifically, the use of a composition or a kit (including aMeltingArray), which comprises the following components (1) and (2)making it possible to discriminate/detect each type of thedisease-causing virus, made it possible to detect a virus causinginfectious aquatic organism disease and discriminate each type of thevirus causing infectious aquatic organism disease:

(1) an oligomer mixture comprising each of PNA probes (SEQ ID NOs: 1 to5) for detection and one or more of primers (SEQ ID NOs: 6 to 10), whichare specific for VHSV, RSIV/ISKNV, RSIV, and KHV, respectively; and

(2) a single-strand generation buffer (SSG buffer) which is used togenerate a single-strand DNA using as a template a PCR amplificationproduct produced using the primer and to hybridize the PNA probe to thesingle-strand DNA.

Herein, the composition or the kit (including MeltingArray) comprisesone or more of the following components (1) to (5) making it possible todiscriminate/detect each type of the virus causing infectious aquaticorganism disease:

(1) an oligomer mixture for detecting a PCR product for identificationof VHSV, in which the oligomer mixture comprises a PNA probe (SEQ IDNO: 1) and a primer (SEQ ID NO: 6);

(2) an oligomer mixture for detecting a “PCR protocol 1(OIE 1)” productfor identification of RSIV/ISKNV, in which the oligomer mixturecomprises a PNA probe (SEQ ID NO: 2) and a primer (SEQ ID NO: 7);

(3) an oligomer mixture for detecting a “PCR protocol 2(OIE 4)” productfor identification of RSIV, in which the oligomer mixture comprises aPNA probe (SEQ ID NO: 3) and a primer (SEQ ID NO: 8);

(4) an oligomer mixture for detecting a “PCR(Bercoiver TK)” product foridentification of KHV, in which the oligomer mixture comprises a PNAprobe (SEQ ID NO: 4) and a primer (SEQ ID NO: 9); and

(5) an oligomer mixture for detecting a “PCR(Gray Sph)” product foridentification of KHV, in which the oligomer mixture comprises a PNAprobe (SEQ ID NO: 5) and a primer (SEQ ID NO: 10).

Therefore, in one aspect, the present invention is directed to a geneticmarker for discrimination or detection of viral hemorrhagic septicemiavirus (VHSV), which is a virus causing infectious aquatic organismdisease, in which the genetic marker is represented by SEQ ID NO: 11.

In another aspect, the present invention is directed to a genetic markerfor discrimination or detection of red sea bream iridovirus (RSIV) orinfectious spleen and kidney necrosis virus (ISKNV), which is a viruscausing infectious aquatic organism disease, in which the genetic markeris represented by SEQ ID NO: 12.

In another aspect, the present invention is directed to a genetic markerfor discrimination or detection of red sea bream iridovirus (RSIV),which is a virus causing infectious aquatic organism disease, in whichthe genetic marker is represented by SEQ ID NO: 13.

In another aspect, the present invention is directed to a genetic markerfor discrimination or detection of Koi herpesvirus (KHV), which is avirus causing infectious aquatic organism disease, in which the geneticmarker is represented by a nucleotide sequence of SEQ ID NO: 14 or SEQID NO: 15.

In still another aspect, the present invention is directed to a primerfor discrimination or detection of viral hemorrhagic septicemia virus(VHSV), which is a virus causing infectious aquatic organism disease, inwhich the primer is represented by a nucleotide sequence of SEQ ID NO:6.

In yet another aspect, the present invention is directed to a primer fordiscrimination or detection of red sea bream iridovirus (RSIV) orinfectious spleen and kidney necrosis virus (ISKNV), which is a viruscausing infectious aquatic organism disease, in which the primer isrepresented by a nucleotide sequence of SEQ ID NO: 7.

In a further aspect, the present invention is directed to a primer fordiscrimination or detection of red sea bream iridovirus (RSIV), which isa virus causing infectious aquatic organism disease, in which the primeris represented by a nucleotide sequence of SEQ ID NO: 8.

In a still further aspect, the present invention is directed to a primerfor discrimination or detection of Koi herpesvirus (KHV), which is avirus causing infectious aquatic organism disease, in which the primeris represented by a nucleotide sequence of SEQ ID NO: 9 or of SEQ ID NO:10.

In a yet further aspect, the present invention is directed to a PNAprobe for discrimination or detection of viral hemorrhagic septicemiavirus (VHSV), which is a virus causing infectious aquatic organismdisease, in which the PNA probe is represented by a nucleotide sequenceof SEQ ID NO: 1.

In another further aspect, the present invention is directed to a PNAprobe for discrimination or detection of red sea bream iridovirus (RSIV)or infectious spleen and kidney necrosis virus (ISKNV), which is a viruscausing infectious aquatic organism disease, in which the primer isrepresented by a nucleotide sequence of SEQ ID NO: 2.

In another further aspect, the present invention is directed to a PNAprobe for discrimination or detection of red sea bream iridovirus(RSIV), which is a virus causing infectious aquatic organism disease, inwhich the primer is represented by a nucleotide sequence of SEQ ID NO:3.

In another further aspect, the present invention is directed to a PNAprobe for discrimination or detection of Koi herpesvirus (KHV), which isa virus causing infectious aquatic organism disease, in which the PNAprobe is represented by a nucleotide sequence of SEQ ID NO: 4 or of SEQID NO: 5.

The PNA probe according to the present invention may have a reporter anda fluorescence quencher attached to both ends thereof. The fluorescencequencher can quench the fluorescence of the reporter. The reporter maybe one or more selected from the group consisting of FAM(6-carboxyfluorescein), Texas red, HEX(2′,4′,5′,7′,-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, Cy3,and Cy5. The quencher may be one or more selected from the groupconsisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 andDabcyl, but is not limited thereto and preferably Dabcyl (FAM-labeled)can be used as the quencher.

Peptide nucleic acid (PNA) is a DNA analogue having nucleic acidconnected by peptide bonds, but not phosphate bonds, and was firstsynthesized by Nielsen et al. in 1991. PNA is artificially synthesizedby a chemical method, but not found in natural systems.

Peptide nucleic acid is one of substances that recognize genes, like LNA(locked nucleic acid) or MNA (morpholino nucleic acid). It isartificially synthesized and has a backbone consisting of polyamide. PNAis greatly excellent in affinity and selectivity and has a highstability for nucleolytic enzyme, and thus is not decomposed by anexisting restriction enzyme. In addition, PNA advantageously has highthermal/chemical properties and stability, and thus its storage is easyand it is not easily broken down.

The PNA forms a duplex by its hybridization to a natural nucleic acidhaving a nucleotide sequence complementary thereto. When they have thesame length, the PNA/DNA duplex is more stable than the DNA/DNA duplexand the PNA/RNA duplex is more stable than the DNA/RNA duplex.Furthermore, since the PNA has a single base mismatch that makes theduplex unstable, the ability of the PNA to detect SNP (single nucleotidepolymorphism) is better than that of natural nucleic acid.

Furthermore, PNA-DNA binding affinity is very high than DNA-DNA bindingaffinity, and thus there is a difference in melting point of about 10 to15° C. even in the presence of one nucleotide mismatch. Using thisdifference in binding affinity, changes in SNP (single-nucleotidepolymorphism) and In/Del nucleotides can be detected.

Although the PNA nucleotide sequence according to the present inventionis not particularly limited, it may be constructed to have a length of12 to 18-mer so as to contain a specific nucleotide sequence (e.g.,nucleotide variation or single nucleotide polymorphism (SNP)) dependingon the kind of virus. In the present invention, a PNA probe may bedesigned to have a desired T_(m) value by adjusting the length of thePNA probe, and even in the case of PNA probes having the same length,the T_(m) value may be adjusted by changing the nucleotide sequence.Furthermore, since a PNA probe has a binding affinity higher than a DNAprobe, it has a higher T_(m) value. Thus, the PNA probe can be designedto have a length shorter than a DNA probe, so that it can detect evenadjacent nucleotide variation or SNP. In a conventional HRM (HighResolution Melt) method, a difference in T_(m) value from a targetnucleic acid is as low as about 0.5° C., and thus an additional analyticprogram or a minute change or correction in temperature is required, andfor this reason, it is difficult to perform analysis, when two or morenucleotide variations or SNPs appear. However, the PNA probe accordingto the present invention is not influenced by the PNA probe sequence andSNP, and thus makes it possible to perform analysis in a simple andconvenient manner.

As described in the present invention, when the PNA probe comprises 14nucleotides, it preferably has a sequence having one or more nucleotidescorresponding to the nucleotide variation or the SNP site of virus, inthe middle of the sequence. Furthermore, the PNA probe may have, in themiddle portion of the nucleotide sequence, a structural modificationincluding a sequence corresponding to the nucleotide variation or theSNP site of virus, thereby further increasing the difference in meltingtemperature (T_(m)) from a target nucleic acid to which it perfectlymatches.

In another still further aspect, the present invention is directed to acomposition and a kit for discrimination or detection of a virus causinginfectious aquatic organism disease, which comprises the above-describedprimer and the above-described PNA probe.

The kit of the present invention may optionally include reagentsrequired for performing a target nucleic acid amplification reaction(e.g., PCR reaction), such as buffer, DNA polymerase cofactor, anddeoxyribonucleotide-5-triphosphate. Alternatively, the kit of thepresent invention may also include various polynucleotide molecules, areverse transcriptase, various buffers and reagents, and an antibodythat inhibits the activities of a DNA polymerase. In addition, in thekit, the optimal amount of the reagent used in a specific reaction canbe easily determined by those skilled in the art who have acquired thedisclosure set forth herein. Typically, the kit of the invention may bemanufactured as a separate package or compartment containing the abovementioned ingredients.

When the kit is used, a single nucleotide mutation and a mutation causedby nucleotide deletion or insertion in a target nucleic acid can beeffectively detected by analysis of a melting curve obtained using thePNA, thereby discriminating viral type.

In still another example of the present invention, for 4 kinds of fishviruses causing infectious aquatic organism diseases, gene nucleotidesequences corresponding to PCR products for detection according to theOIE standards were comparatively analyzed, and based on the results ofthe analysis, a PNA probe represented by each of nucleotide sequences ofSEQ ID NOs: 1 to 5 was hybridized to a single-strand PCR amplificationproduct synthesized using a detection primer represented by each ofnucleotide sequences of SEQ ID NOs: 6 to 10, thereby obtaining meltingcurves. From the melting curves, the melting temperature (Tm) wasdetermined, so that a virus causing infectious aquatic organism viruscould be discriminated and detected.

Therefore, in another yet further aspect, the present invention isdirected to a method for discriminating or detecting a virus causinginfectious aquatic organism disease, the method comprising the steps of:

(a) extracting a target nucleic acid from a sample;

(b) amplifying a genetic marker nucleotide sequence for an infectiousaquatic organism disease-causing virus, contained in the target nucleicacid, by use of a conventional primer pair;

(c) producing a single-strand genetic marker sequence fragment using theamplified genetic marker nucleotide sequence as a template and theabove-described primer;

(d) hybridizing the above-described PNA probe to the producedsingle-strand genetic marker sequence fragment;

(e) obtaining a temperature-dependent melting curve while increasing thetemperature of a PNA probe-hybridized product resulting from step (d);and

(f) discriminating the viral type of the infectious aquatic organismdisease-causing virus or detecting whether or not fish would be infectedwith the viral type by analyzing the melting curve obtained in step (e)to determine a melting temperature.

In yet another example, the present invention may provide a method fordiscriminating or detecting viral hemorrhagic septicemia virus (VHSV),the method comprising the steps of:

(a) extracting a target nucleic acid from a sample;

(b) producing a single-strand genetic marker sequence fragment using agenetic marker nucleotide sequence for viral hemorrhagic septicemiavirus (VHSV) as a template and the primer represented by a nucleotidesequence of SEQ ID NO: 6;

(c) hybridizing a PNA probe represented by a nucleotide sequence of SEQID NO: 1 to the produced single-strand genetic marker sequence fragment;

(d); obtaining a temperature-dependent melting curve while increasingthe temperature of a PNA probe-hybridized product resulting from step(c); and

(e) discriminating or detecting viral hemorrhagic septicemia virus(VHSV) by analyzing the melting curve obtained in step (d) to determinea melting temperature. In yet another example, the present invention mayprovide a method for discriminating or detecting red sea breamiridovirus (RSIV), the method comprising the steps of:

(a) extracting a target nucleic acid from a sample;

(b) producing a single-strand genetic marker sequence fragment using agenetic marker nucleotide sequence for red sea bream iridovirus (RSIV)as a template and the primer represented by a nucleotide sequence of SEQID NO: 8;

(c) hybridizing a PNA probe represented by a nucleotide sequence of SEQID NO: 3 to the produced single-strand genetic marker sequence fragment;

(d); obtaining a temperature-dependent melting curve while increasingthe temperature of a PNA probe-hybridized product resulting from step(c); and

(e) discriminating or detecting red sea bream iridovirus (RSIV) byanalyzing the melting curve obtained in step (d) to determine a meltingtemperature.

In yet another example, the present invention may provide a method fordiscriminating or detecting Koi herpesvirus (HSV) bercoiver TK, themethod comprising the steps of:

(a) extracting a target nucleic acid from a sample;

(b) producing a single-strand genetic marker sequence fragment using agenetic marker nucleotide sequence for Koi herpesvirus (HSV) bercoiverTK as a template and the primer represented by a nucleotide sequence ofSEQ ID NO: 9;

(c) hybridizing a PNA probe represented by a nucleotide sequence of SEQID NO: 4 to the produced single-strand genetic marker sequence fragment;

(d); obtaining a temperature-dependent melting curve while increasingthe temperature of a PNA probe-hybridized product resulting from step(c); and

(e) discriminating or detecting Koi herpesvirus (HSV) bercoiver TK byanalyzing the melting curve obtained in step (d) to determine a meltingtemperature.

In yet another example, the present invention may provide a method fordiscriminating or detecting Koi herpesvirus (HSV) gray Sph, the methodcomprising the steps of:

(a) extracting a target nucleic acid from a sample;

(b) producing a single-strand genetic marker sequence fragment using agenetic marker nucleotide sequence for Koi herpesvirus (HSV) gray Sph asa template and the primer represented by a nucleotide sequence of SEQ IDNO: 10;

(c) hybridizing a PNA probe represented by a nucleotide sequence of SEQID NO: 5 to the produced single-strand genetic marker sequence fragment;

(d); obtaining a temperature-dependent melting curve while increasingthe temperature of a PNA probe-hybridized product resulting from step(c); and

(e) discriminating or detecting Koi herpesvirus (HSV) gray Sph byanalyzing the melting curve obtained in step (d) to determine a meltingtemperature.

In the present invention, the single-strand genetic marker sequencefragment may be produced by adding a single strand generation buffer(SSG buffer).

In the present invention, the single strand generation buffer maycomprise DNA polymerase, dNTPs (deoxynucleotides) and a stabilizer, inwhich the DNA polymerase may be Taq polymerase, but is not limitedthereto.

In the present invention, step (b) of amplifying the genetic markernucleotide sequence by use of the conventional primer pair may furthercomprise adding a TaqMan probe to obtain an amplification curve.

In the present invention, when two or more target nucleic acids are usedand the reporter attached to the PNA probe is changed depending on thekind of target nucleic acid, the viral type of one or more virusescausing infectious aquatic organism disease can be discriminated ordetected by detecting two or more target nucleic acids.

In the present invention, the amplification may be performed by areal-time PCR (polymerase chain reaction) method.

As used herein, the term “sample” is meant to include various samples.Preferably, a biosample is analyzed using the method of the presentinvention. More preferably, the sample may be either a sample that ismixed with the viralspecies, or a sample from an individual (forexample, fish or the like) infected with the virus. Biosamplesoriginated from plants, animals, humans, fungi, bacteria and virus canbe analyzed. When a mammal- or human-originated sample is analyzed, itmay be derived from specific tissues or organs. Representative examplesof tissues include connective, skin, muscle, or nervous tissue.Representative examples of organs include eyes, brain, lung, liver,spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage,pancreas, kidney, gallbladder, stomach, small intestine, testis, ovary,uterus, rectum, nervous system, and gland and internal blood vessels. Abiosample to be analyzed includes any cell, tissue or fluid that isderived from a biological origin, or any other medium that can be wellanalyzed by the present invention. The biosample also includes a sampleobtained from foods produced for consumption of humans and/or animals.In addition, the to-be-analyzed biosample includes a body fluid sample,which includes, but not limited to, blood, serum, plasma, lymph, breastmilk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g.,pulverized brain), spinal fluid, appendix, spleen, and tonsil tissueextracts.

As used herein, the term “target nucleic acid”, “synthetic DNA” or“artificially synthesized oligo” means a nucleic acid sequence(containing SNP or nucleotide variation) to be detected. The targetnucleic acid comprises a specific region of the nucleic acid sequence ofa “target gene” encoding a protein having physiological and biochemicalfunctions, and is annealed or hybridized to the primer or the probeunder hybridization, annealing or amplification conditions.

As used herein, the term “hybridization” means that complementarysingle-stranded nucleic acids form a double-stranded nucleic acid.Hybridization can occur when the complementarity between two nucleicacid strands is perfect (perfect match) or when some mismatched residuesexist. The degree of complementarity necessary for hybridization mayvary depending on hybridization conditions, particularly may becontrolled by temperature.

In the present invention, the melting curve analysis may be performed bya fluorescence melting curve analysis (FMCA) method.

The PNA probe comprising the reporter and the quencher according to thepresent invention generates a fluorescent signal after its hybridizationto the target nucleic acid. As the temperature increases, the PNA probeis rapidly melted with the target nucleic acid at its suitable meltingtemperature, and thus the fluorescent signal is quenched. Throughanalysis of a high-resolution melting curve obtained from thefluorescent signal as a function of this temperature, the presence orabsence of a nucleotide modification (including nucleotide variation orSNP) may be detected. If the PNA probe perfectly matches with thenucleotide sequence of the target nucleic acid, it then shows anexpected melting temperature (T_(m)) value, but if the PNA probemismatches with a target nucleic acid in which a nucleotide mutation ispresent, it shows a melting temperature (T_(m)) value lower than anexpected value.

As used herein, the term “nucleotide variation” refers to a change in anucleotide sequence of a target nucleic acid (e.g., a substitution,deletion or insertion of one or more nucleotides, as well as a singlenucleotide polymorphism (SNP)) relative to a reference sequence. The PNAprobe of the present invention can analyze a change in a nucleotidesequence of a target nucleic acid, including SNP of the target nucleicacid or a substitution, deletion or insertion of nucleotides of thetarget nucleic acid through the melting curve analysis.

The PNA probe according to the present invention may have a reporter anda fluorescence quencher attached to both ends. The fluorescence quenchercan quench the fluorescence of the reporter. The reporter may be one ormore selected from the group consisting of FAM (6-carboxyfluorescein),Texas red, HEX(2′,4′,5′,7′,-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, Cy3,and Cy5. The quencher may be one or more selected from the groupconsisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 andDabcyl, but is not limited thereto and preferably Dabcyl (FAM-labeled)can be used as the quencher.

The T_(m) value also changes depending on the difference between thenucleotide sequence of the PNA probe and the nucleotide sequence of aDNA complementary thereto, and thus the development of applicationsbased on this change is easily achieved. The PNA probe is analyzed usinga hybridization method different from a method for hybridization of aTaqMan probe, and probes having functions similar to that of the PNAprobe include molecular beacon probes and scorpion probes.

A specific nucleotide sequence (e.g., nucleotide variation or SNP)analysis using the PNA probe can be sufficiently achieved using aforward/reverse primer set (according to Office of InternationalEpizootics (OIE) standards for the conventional primer pair) for PCR, aprobe comprising nucleotide(s) that recognize(s) the specific nucleotidesequence, and a primer of producing a single-strand genetic markersequence fragment using a genetic marker nucleotide sequence amplifiedby the primer set as a template. The PCR may be performed using aconventional method, and after completion of the PCR, a melting processis required. Whenever the melting temperature increases by 0.5° C., theintensity of fluorescence is measured to obtain the T_(m) value. Inparticular, general real-time PCR systems are widely known and have anadvantage in that purchase of an additional program such as a HRM(high-resolution melting) program or a minute temperature change is notrequired.

Melting curve analysis according to the present invention is a method ofanalyzing a double-chain nucleic acid formed of the target nucleic acidDNA or RNA and the probe. This method is called “melting curveanalysis”, because it is performed by, for example, T_(m) analysis orthe analysis of the melting curve of the double-strand nucleic acid.Using a probe complementary to a specific nucleotide sequence (includingnucleotide variation or SNP) of a target to be detected, a hybrid(double-chain DNA) of a target single-chain DNA and the probe is formed.Subsequently, the formed hybrid is heated, and the dissociation(melting) of the hybrid, which results from an increase in thetemperature, is detected based on a change in a signal such asabsorbance. Based on the results of the detection, the T_(m) value isdetermined, thereby determining the presence or absence of the specificnucleotide sequence. The T_(m) value increases as the homology of theformed hybrid increases, and the T_(m) value decreases as the homologydecreases. For this reason, the T_(m) value of a hybrid formed of aspecific nucleotide sequence of a target to be detected and a probecomplementary thereto is previously determined (a reference value forevaluation), and the T_(m) value of a hybrid formed of the targetsingle-chain DNA of a sample to be detected and the probe is measured (ameasured value). If the measured value is approximately equal to thereference value, it can be determined that the probe matches, that is, aspecific nucleotide sequence is present in the target DNA. If themeasured value is lower than the reference value, the probe mismatches,that is, no mutation is present in the target DNA.

The fluorescent melting curve analysis of the present invention is amethod that analyzes a melting curve using a fluorescent material, andmore specifically, may analyze the melting curve by using a probecontaining a fluorescent material. The fluorescent material may beeither a reporter or a quencher, and may preferably be an intercalatingfluorescent material.

In the real-time polymerase chain reaction (PCR) method according to thepresent invention, a fluorescent substance is intercalated into adouble-stranded DNA duplex during PCR, and the temperature is increasedtogether with amplification to melt the DNA double strands to therebyreduce the amount of fluorescent substance present between the DNAdouble strands. The resulting melting curve pattern, particularly thetemperature (T_(m)) at which the DNA is melted (denatured), may beanalyzed, thereby detecting and/or discriminating the type of virusbased on the presence or absence of the specific nucleotide sequence(including nucleotide variation or SNP).

FIG. 1 or 2 is a conceptual view illustrating the technicalcharacteristics of MeltingArray for PCR verification according to oneembodiment of the present invention. As shown therein, a PNA probe mayhybridize to a target nucleic acid, and then generate a fluorescencesignal. As the temperature increases, the PNA probe is rapidly meltedout from the target nucleic acid at its suitable melting temperature(T_(m)), and thus the fluorescent signal is quenched. According to thepresent invention, through high-resolution fluorescence melting curveanalysis (FMCA) obtained from the fluorescent signal as a results ofthis temperature change, the presence or absence of a target nucleicacid and a difference in the nucleotide sequence may be detected. If thePNA probe according to the present invention perfectly matches with thenucleotide sequence of the target nucleic acid, it then shows anexpected melting temperature (T_(m)) value, but if the PNA probemismatches with a target nucleic acid in which a nucleotide mutation ispresent, it then shows a melting temperature (T_(m)) value lower than anexpected value. If there is no target nucleic acid, the PNA probe thenshows no melting temperature (T_(m)) value.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are illustrative purposesonly and are not to be construed to limit the scope of the presentinvention.

Example 1: Construction of Genetic Markers for Discrimination andDetection of Viruses Causing Infectious Aquatic Organism Diseases, andPrimers and PNA Probes Specific for the Viruses

1-1: Virus Causing Infectious Aquatic Organism Disease: VHSV

The sequence of a gene fragment, targeting the N-gene of VHSV andsynthesized by “PCR method for detection according to the OIEstandards”, was analyzed comparatively with the nucleotide sequenceregistered in the nucleotide database (DB) of the National Center forBiotechnology Information (NCBI) in order to obtain a gene nucleotidesequence for each type of virus.

As a result, the common nucleotide sequence of VHSV represented by5′-GACATGGGCTTCA-3′ (SEQ ID NO: 11) was obtained, and the nucleotidesequence was selected as a genetic marker for discrimination ordetection of VHSV.

Furthermore, as a primer for production of a single-strand DNA for thegenetic marker, primer 1 represented by SEQ ID NO: 6 was constructed,and as a probe for hybridization to the genetic marker, PNA 1represented by SEQ ID NO: 1 was constructed.

FIG. 3 is a nucleotide sequence view illustrating the nucleotidesequence of a portion of the gene of VHSV for discrimination/detectionand an example of the nucleotide sequence of PNA obtained therefrom. InFIG. 3, the nucleotide sequence corresponding to the PNA probe isindicated by blue color.

1-2: Virus Causing Infectious Aquatic Organism Disease: RSIV/ISKNV

The sequence of a gene fragment of RSIV/ISKNV, synthesized by “PCRprotocol 1 (OIE 1) method” for detection according to the OIEstandards”, was analyzed comparatively with the nucleotide sequenceregistered in the nucleotide database (DB) of the National Center forBiotechnology Information (NCBI) in order to obtain a gene nucleotidesequence for each type of virus.

As a result, the common nucleotide sequence of RSIV/ISKNV represented by5′-CCATGTACAACATGCTC-3′ (SEQ ID NO: 12) was obtained, and the nucleotidesequence was selected as a genetic marker for discrimination ordetection of RSIV/ISKNV.

Furthermore, as a primer for production of a single-strand DNA for thegenetic marker, primer 2 represented by SEQ ID NO: 7 was constructed,and as a probe for hybridization to the genetic marker, PNA 2represented by SEQ ID NO: 2 was constructed.

FIG. 4 is a nucleotide sequence view illustrating the nucleotidesequence of a portion of the gene of RSIV/ISKNV fordiscrimination/detection and an example of the nucleotide sequence ofPNA obtained therefrom. In FIG. 4, the nucleotide sequence correspondingto the PNA probe is indicated by blue color.

1-3: Virus Causing Infectious Aquatic Organism Disease: RSIV

The sequence of a gene fragment of RSIV, synthesized by “PCR protocol 2(OIE 4) method” for detection according to the OIE standards”, wasanalyzed comparatively with the nucleotide sequence registered in thenucleotide database (DB) of the National Center for BiotechnologyInformation (NCBI) in order to obtain a gene nucleotide sequence foreach type of virus.

As a result, the common nucleotide sequence of RSIV/ISKNV represented by5′-CCAAGTTCATCATC-3′ (SEQ ID NO: 13) was obtained, and the nucleotidesequence was selected as a genetic marker for discrimination ordetection of RSIV.

Furthermore, as a primer for production of a single-strand DNA for thegenetic marker, primer 3 represented by SEQ ID NO: 8 was constructed,and as a probe for hybridization to the genetic marker, PNA 3represented by SEQ ID NO: 3 was constructed.

FIG. 5 is a nucleotide sequence view illustrating the nucleotidesequence of a portion of the gene of RSIV for discrimination/detectionand an example of the nucleotide sequence of PNA obtained therefrom. InFIG. 5, the nucleotide sequence corresponding to the PNA probe isindicated by blue color.

1-4: Virus Causing Infectious Aquatic Organism Disease: KHV

The sequence of a gene fragment of KHV, synthesized by “Bercoiver TK PCRmethod” for detection according to the OIE standards”, was analyzedcomparatively with the nucleotide sequence registered in the nucleotidedatabase (DB) of the National Center for Biotechnology Information(NCBI) in order to obtain a gene nucleotide sequence for each type ofvirus.

As a result, the common nucleotide sequence of KHV represented by5′-GTTCTTCCCCGAC-3′ (SEQ ID NO: 14) was obtained, and the nucleotidesequence was selected as a genetic marker for discrimination ordetection of KHV.

Furthermore, as a primer for production of a single-strand DNA for thegenetic marker, primer 4 represented by SEQ ID NO: 9 was constructed,and as a probe for hybridization to the genetic marker, PNA 4represented by SEQ ID NO: 4 was constructed.

FIG. 6 is a nucleotide sequence view illustrating the nucleotidesequence of a portion of the gene of KHV for discrimination/detectionand an example of the nucleotide sequence of PNA obtained therefrom. InFIG. 6, the nucleotide sequence corresponding to the PNA probe isindicated by blue color.

1-5: Virus Causing Infectious Aquatic Organism Disease: KHV

The sequence of a gene fragment of KHV, synthesized by “Gray Sph PCRmethod” for detection according to the OIE standards”, was analyzedcomparatively with the nucleotide sequence registered in the nucleotidedatabase (DB) of the National Center for Biotechnology Information(NCBI) in order to obtain a gene nucleotide sequence for each type ofvirus.

As a result, the common nucleotide sequence of KHV represented by5′-TCTCAGCAACACC-3′ (SEQ ID NO: 15) was obtained, and the nucleotidesequence was selected as a genetic marker for discrimination ordetection of KHV.

Furthermore, as a primer for production of a single-strand DNA for thegenetic marker, primer 5 represented by SEQ ID NO: 10 was constructed,and as a probe for hybridization to the genetic marker, PNA 5represented by SEQ ID NO: 5 was constructed.

FIG. 7 is a nucleotide sequence view illustrating the nucleotidesequence of a portion of the gene of KHV for discrimination/detectionand an example of the nucleotide sequence of PNA obtained therefrom. InFIG. 7, the nucleotide sequence corresponding to the PNA probe isindicated by blue color.

As a result, the nucleotide sequences of the viral genetic marker, PNAprobe and primer according to the present were determined as shown inTable 1 below.

TABLE 1 Sequences Name SEQ ID NO: (5′→3′) Modification Target PNA PNA 1SEQ ID NO: 1 TGAAGCCCATGTC TexasRed, Dabsy1 VHSV probe PNA 2SEQ ID NO: 2 CCATGTACAACATGC RSIV, ISKNV TC PNA 3 SEQ ID NO: 3GATGATGAACTTGG RSIV PNA 4 SEQ ID NO: 4 GTTCTTCCCCGAC KHV Bercoiver TKPNA 5 SEQ ID NO: 5 TCTCAGCAACACC KHV Gray Sph Primers primer 1SEQ ID NO: 6 ATGGAAGGAGGAATT — VHSV CGTGAAGCG primer 2 SEQ ID NO: 7GCACCAACACATCTC — RSIV, ISKNV CTATC primer 3 SEQ ID NO: 8CGGGGGCAATGACGA — RSIV CTACA primer 4 SEQ ID NO: 9 CACCCAGTAGATTAT —KHV Bercoiver TK GC primer 5 SEQ ID NO: 10 GACACATGTTACAAT —KHV Gray Sph GGTCGC Viral marker VM 1 SEQ ID NO: 11 GACATGGGCTTCA — VHSVVM 2 SEQ ID NO: 12 CCATGTACAACATGC — RSIV, ISKNV TC VM 3 SEQ ID NO: 13CCAAGTTCATCATC — RSIV VM 4 SEQ ID NO: 14 GTTCTTCCCCGAC — KHV VM 5SEQ ID NO: 15 TCTCAGCAACACC — KHV

TaqMan and PNA probes were labeled with FAM, HEX, TexasRed and Cy5,respectively, such that they would not contain the same fluorescence.Then, PNA probes were constructed using the nucleotide sequence as shownin Table 1 above, a reporter and a quencher. The PNA probes used in thepresent invention were designed using a PNA probe designer (AppliedBiosystems, USA), and the PNA probes were synthesized using a HPLCpurification method by Panagene (Korea). The purities of all thesynthesized probes were analyzed by mass spectrometry (the unnecessarysecondary structures of the probes were avoided for effective binding totarget nucleic acids).

Example 2: Optimization of MeltingArray Kit for Discrimination orDetection of Virus Causing Infectious Aquatic Organism Disease

Using PNA probes and primers constructed in Example 1, amplificationcurves and melting curves for DNA samples of four kinds of virusescausing infectious aquatic organism diseases were obtained and analyzedto verify PCR products, thereby optimizing the discrimination ordetection of the disease-causing viruses. Herein, a TaqMan probe (and aconventional primer pair corresponding thereto) for discrimination ordetection of the disease-causing viruses according to the OIE (Office ofInternational Epizootics) standards may be used.

A MeltingArray reaction was performed using a CFX96™ real-time system(BIO-RAD, USA). To produce a single-strand target nucleic acid from thePCR product, a single-strand generation buffer (SSG buffer) and a singleprimer complementary to the binding strand of the probe were used. Thecomposition of the SSG buffer comprised 2×nTaq-HOT (0.2 units/μl),nTaq-HOT buffer (containing 4 mM MgCl₂), a dNTP mixture (A, T, G and C;0.4 mM for each) and a stabilizer.

The composition of reactants for the MeltingArray reaction is shown inTable 2 below. A master mix for a MeltingArray kit was prepared, andthen 1 to 3 μL of the PCR product was added thereto, followed byanalysis.

TABLE 2 Composition Content 2X SSG buffer 10 μL Oligomer mix (PNA probe,primer) 1.5 μL Template(Template, PCR product) 1~3 μL Distilled water upto 20 μL

Table 3 shows conditions for hybridization reaction of reactants.Specifically, Table 3 shows a step of producing a single-strand DNA fromthe PCR product, a denaturation step, and a process of hybridizing thePNA probe and increasing the temperature of the hybridized product.

TABLE 3 Temper- Steps ature(° C.) Reaction time and cycle Single strand95 5-10 min   generation) 95 30 sec 15-20 cycle 56 30 sec 76 30 secDenaturation 95  1 min Probe binding 75 30 sec  55 30 sec  Melting 45 to80 Increment 1.0° C., 5 sec (TexasRed)

As a result, as can be seen in FIG. 8, viruses causing infectiousaquatic organism diseases could be discriminated or detected by analysisof melting curves obtained using five kinds of PNA probes.

Example 3: Method for Discriminating or Detecting Virus Based on MeltingPeak Obtained Using PNA Probe

When the viral type for a unknown viral DNA sample is to bediscriminated or detected using the PNA probes according to the presentinvention, a table listing scores at different melting temperatures asshown in Table 4 below can be previously prepared and can be used.

After melting curve analysis was performed as described in Example 2,the obtained fluorescence signal and T_(m) value were digitizedaccording to the temperature at which a perfect match appeared.Specifically, the range of perfect match temperature±2° C. is made, andwhen the T_(m) value for a unknown viral DNA sample is within thisrange, the type of virus in the viral sample can be identified anddiscriminated.

TABLE 4 Fluorescent material of PNA probe Tm(° C.) Kind of detectablevirus TexasRed 65 VHSV 65 RSIV, ISKNV 66 RSIV 60 KHV 62 KHV

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof

What is claimed is:
 1. A PNA probe consisting of SEQ ID NO: 2, fordiscrimination or detection of a virus causing red sea bream iridovirusdisease which is a virus causing infectious aquatic organism disease,wherein the virus causing red sea bream iridovirus disease is red seabream iridovirus (RSIV) or infectious spleen and kidney necrosis virus(ISKNV).
 2. A PNA probe consisting of SEQ ID NO: 2 with one or more of areporter and a quencher attached thereto, wherein the probe is fordiscrimination or detection of a virus causing red sea bream iridovirusdisease which is a virus causing infectious aquatic organism disease. 3.A composition for discrimination or detection of a virus causing red seabream iridovirus disease, the composition comprising: a primerconsisting of SEQ ID NO: 7 and a PNA probe consisting of SEQ ID NO: 2.4. A kit for discrimination or detection of a virus causing red seabream iridovirus disease, the kit comprising: a primer consisting of SEQID NO: 7; and a PNA probe consisting of SEQ ID NO:
 2. 5. A method fordetecting a virus causing red sea bream iridovirus disease, the methodcomprising the steps of: (a) extracting a target nucleic acid from afish sample; (b) amplifying a genetic marker nucleotide sequence for thevirus causing red sea bream iridovirus disease contained in the targetnucleic acid by use of a primer pair capable of amplifying a fragmentconsisting of SEQ ID NO: 17 or SEQ ID NO: 18; (c) producing asingle-strand genetic marker sequence fragment using the amplifiedgenetic marker nucleotide sequence as a template and a primer consistingof SEQ ID NO: 7; (d) hybridizing a PNA probe consisting of a reporterand quencher labeled SEQ ID NO: 2 to the produced single-strand geneticmarker sequence fragment; (e) obtaining a temperature-dependent meltingcurve while increasing the temperature of a PNA probe-hybridized productresulting from step (d); and (f) detecting whether or not the fishsample is infected with the virus causing red sea bream iridovirusdisease by analyzing the melting curve obtained in step (e) to determinea melting temperature, wherein the virus causing red sea breamiridovirus disease is red sea bream iridovirus (RSIV) or infectiousspleen and kidney necrosis virus (ISKNV).
 6. The method of claim 5, thesingle-strand genetic marker sequence fragment in step (c) is producedby adding a single strand generation buffer (SSG buffer).
 7. The methodof claim 6, wherein the single strand generation buffer comprises DNApolymerase, dNTPs (deoxynucleotides) and a stabilizer.
 8. The method ofclaim 5, wherein step (b) of amplifying the genetic marker nucleotidesequence by use of the primer pair further comprises adding a TaqManprobe to obtain an amplification curve.